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Conditional Knockdown of Proteins Using Auxin-inducible Degron (AID) Fusions in Toxoplasma gondii
在刚地弓形虫中通过融合生长素诱导的降解因子(AID)进行蛋白质条件性敲减   

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本实验方案简略版
PLOS Pathogens
May 2017

Abstract

Toxoplasma gondii is a member of the deadly phylum of protozoan parasites called Apicomplexa. As a model apicomplexan, there is a great wealth of information regarding T. gondii’s 8,000+ protein coding genes including sequence variation, expression, and relative contribution to parasite fitness. However, new tools are needed to functionally investigate hundreds of putative essential protein coding genes. Accordingly, we recently implemented the auxin-inducible degron (AID) system for studying essential proteins in T. gondii. Here we provide a step-by-step protocol for examining protein function in T. gondii using the AID system in a tissue culture setting.

Keywords: Auxin (生长素), Degron (降解因子), AID (AID), Conditional knockdown (条件性敲减), Protein regulation (蛋白质调节), Parasite (寄生虫), Toxoplasma gondii (刚地弓形虫), CRISPR (CRISPR)

Background

Auxins are a class of phytohormones that signal by targeting certain proteins for proteasomal degradation in plants (Teale et al., 2006). Kohei Nishimura et al. had the clever idea of transferring components of this plant-specific signaling system to other eukaryotes for conditional regulation of proteins of interest (POIs), creating the auxin-inducible degron (AID) system (Nishimura et al., 2009). This system has since been adapted successfully in several eukaryotes, including the apicomplexan parasite Plasmodium (Kreidenweiss et al., 2013; Philip and Waters, 2015). Just two transgenic components are needed to implement this system, a plant auxin receptor called transport inhibitor response 1 (TIR1) and a POI tagged with an AID. Treatment with an auxin (e.g., 3-indolacetic acid/IAA) activates the SCFTIR1 ubiquitin ligase complex which exclusively targets AID-tagged proteins for ubiquitin-dependent proteasomal degradation (Figure 1). We recently engineered an RHΔhxgprtΔku80 line of T. gondii to stably express TIR1 from Oryza sativa (RH TIR1-3FLAG) (Brown et al., 2017; Long et al., 2017a). In this background, we were able to use CRISPR/Cas9 genome editing (Shen et al., 2014; Sidik et al., 2014; Shen et al., 2017) to tag essential T. gondii genes of interest with AID-3HA or mini-AID(mAID)-3HA, regulate their expression with auxin, and identify phenotypes associated with their loss (Brown et al., 2017; Long et al., 2017a and 2017b).

There are several advantages for using this system for conditional knockdowns in T. gondii. First, POI-AID fusions are expressed from their endogenous promoters, maintaining normal expression timing and levels. Second, auxin is non-toxic to parasite and host cell cultures at 1 mM but can function as low as ~50 µM. Third, auxin is added only when knockdown is desired and is commercially available for less than $5 USD per gram. Last and most importantly, POI-AID fusions are fully degraded in as little as 15 min following auxin treatment. For these reasons, we were compelled to elaborate on our published methods in this detailed protocol to facilitate the establishment of this system in other apicomplexan laboratories.


Figure 1. Model of the auxin inducible-degron system. A. In the absence of auxin, the plant auxin receptor TIR1 is in its inactive ‘Apo’ state, allowing the protein of interest (POI)-AID-3HA fusion to express and function normally. B. Auxin-bound TIR1 assembles into an active Skp-Cullen-F Box (SCFTIR1) ubiquitin ligase complex where it recognizes and polyubiquitinates AID. C. The polyubiquitin modification targets POI-AID-3HA for proteasomal degradation.

Materials and Reagents

  1. Microcentrifuge tubes (1.7 ml)
  2. PCR tubes (0.2 ml)
  3. T-25 and T-175 culture flasks (Corning, catalog numbers: 430639 , 431080 )
  4. 96-well tissue culture plates (TPP, catalog number: 92696 )
  5. 24-well plates (TPP, catalog number: 92024 )
  6. 22 G blunt needles (CML Supply, catalog number: 901-22-100M )
  7. 3.0 µm pore size 47 mm filter membrane (GE Healthcare, Whatman, catalog number: 111112 )
  8. 10 ml syringes (BD, catalog number: 309695 )
  9. 50 ml polystyrene conical vials (Fisher Scientific, catalog number: 05-539-10 )
  10. 1 L Stericup Filter Units (Merck, catalog number: SCVPU11RE )
  11. 13 mm cell scrapers (TPP, catalog number: 99002 )
  12. 47 mm polycarbonate syringe filter holder (GE Healthcare, Whatman, catalog number: 420400 )
  13. Filter paper for Western blot wet transfer (GE Healthcare, Whatman, catalog number: 3030-917 )
  14. Nitrocellulose membrane (GE Healthcare, Amersham, catalog number: 10600003 )
  15. Petri dishes (Sigma-Aldrich, catalog number: P5606 )
  16. Pipette tips for Gilson pipettes (Gilson, catalog numbers: F171101 , F171301 , F171501 )
  17. Sterile serological pipettes (5 ml, 10 ml, 25 ml)
  18. Electroporation cuvettes 4 mm gap (BTX, catalog number: 45-0126 )
  19. pSAG1::Cas9-U6::sgUPRT plasmid (Addgene, catalog number: 54467 ) (Shen et al., 2014)
  20. pYFP-AID-3HA, Floxed HXGPRT plasmid (Addgene, catalog number: 87260 ) (Long et al., 2017a)
  21. pYFP-mAID-3HA, Floxed HXGPRT plasmid (Addgene, catalog number: 87259 ) (Brown et al., 2017)
  22. T. gondii line RH TIR1-3FLAG (genotype: RHΔhxgprtΔku80; TUB1:TIR1-3FLAG, SAG1:CAT) (Brown et al., 2017; Long et al., 2017a)
  23. NEB5α chemically-competent E. coli with SOC medium (New England Biolabs, catalog number: C2987I )
  24. Human foreskin fibroblasts (HFF) (ATCC, catalog number: SCRC-1041 )
  25. Q5 Site-Directed Mutagenesis Kit with chemically competent E. coli (New England Biolabs, catalog number: E0554S )
  26. Mutagenesis primers for reprogramming pSAG1::Cas9-U6::sgUPRT (IDT, 25 nmole, standard desalting)
  27. 2x SDS-PAGE sample buffer (Sigma-Aldrich, catalog number: S3401 )
  28. 1 kb DNA ladder (New England Biolabs, catalog number: N3232 )
  29. Agarose (Fisher Scientific, catalog number: BP160 )
  30. SDS-PAGE 4-15% gradient Tris-glycine polyacrylamide gels (Bio-Rad Laboratories, catalog number: 4561086 )
  31. 6x Gel Loading Dye (New England Biolabs, catalog number: B7025 )
  32. LB broth (BD, catalog number: 244610 )
  33. Ampicillin (Sigma-Aldrich, catalog number: A9518 )
  34. Plasmid miniprep kit (Macherey-Nagel, catalog number: 740588 )
  35. M13 Reverse universal primer (5’-ACAGGAAACAGCTATGAC) (Genewiz)
  36. Q5 DNA Polymerase (New England Biolabs, catalog number: M0491 )
  37. dNTPs 10 mM each (New England Biolabs, catalog number: N0447 )
  38. Gene of interest tagging primers for amplifying (m)AID-3HA, Floxed HXGPRT tagging cassette with short homology flanks (IDT, 25 nmole, standard desalting)
  39. Gene of interest diagnostic tagging primers (IDT, 25 nmole)
  40. Agarose Gel and PCR Cleanup Kit (Macherey-Nagel, catalog number: 740609 )
  41. Trypsin-EDTA (Sigma-Aldrich, catalog number: T3924 )
  42. ATP (Sigma-Aldrich, catalog number: A6419 )
  43. Glutathione (Sigma-Aldrich, catalog number: G6013 )
  44. Mycophenolic acid (Sigma-Aldrich, catalog number: M3536 )
  45. Xanthine (Sigma-Aldrich, catalog number: X4002 )
  46. Proteinase K (Sigma-Aldrich, catalog number: P2308 )
  47. Taq polymerase (New England Biolabs, catalog number: M0273 )
  48. Ethanol (EtOH) (Pharmco-AAPER, catalog number: 11100020 )
  49. GelRed nucleic acid stain (Biotium, catalog number: 41001 )
  50. Licor anti-mouse 800CW secondary antibody (LI-COR, catalog number: 925-32210 )
  51. Licor anti-rabbit 680RD secondary antibody (LI-COR, catalog number: 925-68071 )
  52. Mouse anti-HA monoclonal antibody (BioLegend, catalog number: 901501 )
  53. Non-fat powdered milk (Nestle Carnation)
  54. Rabbit anti-Aldolase (T. gondii) (Starnes et al., 2009) or other T. gondii loading control antibody
  55. Tris base (Sigma-Aldrich, catalog number: T6066 )
  56. Glacial acetic acid (Fisher Scientific, catalog number: A38-500 )
  57. 0.5 M EDTA pH 8.0 (Merck, catalog number: 324504 )
  58. Boric acid (Sigma-Aldrich, catalog number: B6768 )
  59. Glycine (Sigma-Aldrich, catalog number: G7128 )
    Note: This product has been discontinued.
  60. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L5750 )
  61. Methanol (Fisher Scientific, catalog number: A412P )
  62. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P5405 )
  63. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271 )
  64. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
  65. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P8281 )
  66. Tween-20 (Sigma-Aldrich, catalog number: P2287 )
  67. Dulbecco’s Modified Eagle’s Medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 12100046 )
  68. Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
  69. 200 mM L-glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030149 )
  70. 10 mg/ml gentamicin (Thermo Fisher Scientific, GibcoTM, catalog number: 15710072 )
  71. Characterized fetal bovine serum (FBS) (GE Healthcare, catalog number: SH30071.01HI )
  72. Hanks’ balanced salt solution (Sigma-Aldrich, catalog number: H9269 )
  73. HEPES (Sigma-Aldrich, catalog number: H3375 )
  74. EGTA (Merck, catalog number: 324626 )
  75. EDTA (Merck, catalog number: 324504 )
  76. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  77. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M4880 )
  78. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
  79. 3-indoleacetic acid (IAA/auxin) (Sigma-Aldrich, catalog number: I2886 )
  80. Agar (Fisher Scientific, catalog number: BP1423 )
  81. 10x Tris-Acetate-EDTA (TAE) buffer (see Recipes)
  82. 10x Tris-Borate-EDTA (TBE) buffer (see Recipes)
  83. 10x SDS-PAGE running buffer (see Recipes)
  84. 10x protein transfer buffer (see Recipes)
  85. 1x protein transfer buffer (see Recipes)
  86. 10x phosphate buffered saline (PBS) (see Recipes)
  87. Phosphate buffered saline + Tween-20 (PBST) (see Recipes)
  88. PCR lysis buffer (see Recipes)
  89. D10 medium (see Recipes)
  90. Hank’s balanced salt solution with HEPES and EGTA (HHE) (see Recipes)
  91. 0.1 M KPO4 buffer pH 7.6 (for Cytomix buffer) (see Recipes)
  92. Cytomix electroporation buffer pH 7.6 (Soldati and Boothroyd, 1993) (see Recipes)
  93. 500 mM 3-indoleacetic acid (IAA/auxin) (1,000x Stock) (see Recipes)

Equipment

  1. Autoclave (Steris, model: SG-120 )
  2. Benchtop centrifuge with 15 ml and 50 ml conical vial holders (Eppendorf, model: 5810 R )
  3. Biological safety cabinet (Baker, model: SterilGuard® II )
  4. CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM 310 )
  5. Detergent free pyrex glassware and stir bars for media preparation
  6. Electroporator (BTX, model: ECM 830 )
  7. Gel documentation system (Bio-Rad Laboratories, model: Gel DocTM XR+ )
  8. Incubating orbital shaker (VWR, model: Model 3500I )
  9. Inverted phase contrast microscope (Nikon Instruments, model: Eclipse TS100 )
  10. Hemacytometer (Sigma-Aldrich, model: Bright-LineTM )
  11. Licor Odyssey imaging system (LI-COR, model: Odyssey® CLx )
  12. Microcentrifuge (Eppendorf, model: 5417 R )
  13. Microvolume spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM One )
  14. Microwave (VWR, catalog number: 75856-526)
    Manufacturer: Argos Technologies, catalog number: 111092 .
  15. Pipet aid (Thermo Fisher Scientific, Thermo ScientificTM, model: S1 )
  16. Pipettes (Pipetman, Gilson, models: P2 , P20 , P200 , P1000 )
  17. Protein wet transfer blotting apparatus (Bio-Rad Laboratories, model: Mini Trans-Blot® )
  18. Refrigerator Freezer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 10ECEETSA )
  19. SDS-PAGE electrophoresis apparatus (Bio-Rad Laboratories, model: Mini-Protean Tetra Cell )
  20. Standard orbital shaker (VWR, model: Model 1000 )
  21. Thermal cycler (Thermo Fisher Scientific, Applied BiosystemsTM, model: VeritiTM 96-well )

Software

  1. Google Chrome (Google) or other web browsing software
  2. Snapgene (Snapgene) or other plasmid viewing software
  3. Image studio lite (LI-COR) or other gel analysis software
  4. Axiovision (Carl Zeiss Microscopy) or other cellular imaging software
  5. ImageJ (Developed by Wayne Rasband) or other image quantification software
  6. GraphPad Prism (GraphPad Software Inc.) or other graphing and statistical analysis software

Procedure

  1. Generating gene-specific Cas9/sgRNA plasmids for C-terminal tagging
    1. Genomic sequence retrieval
      1. From www.toxodb.org, navigate to the gene of interest (GOI)’s gene page (Figure 2A). In general GT1 sequences are used when working with the RH TIR1-3FLAG line. Click the ‘Download Gene’ link above the Gene ID (Figure 2A).
      2. From the ‘Download Gene’ page on ToxoDB.org, download the 3’ tagging region by selecting the following options: Choose a Report: FASTA; Type of sequence: Genomic; Region: Translation stop -80, Translation stop +200; Download type: Show in browser. Select ‘Get Sequences’ link (Figure 2B).
      3. From the sequence retrieval page, select the genomic sequence without the FASTA identifiers and copy to clipboard (Figure 2C).
    2. Protospacer selection
      1. From the Eukaryotic Pathogen CRISPR guide RNA/DNA Design Tool webpage (EuPaGDT, http://grna.ctegd.uga.edu/), begin by entering a ‘Job Name’. Use the default settings recommended for SpCas9: ’20 nt gRNA NGG PAM’. Under the genome selection tab, select the T. gondii lineage and database (e.g., T. gondii GT1 ToxoDB-32). Paste the copied genomic sequence from ToxoDB.org and select ‘Get guide RNA’ (Figure 2D).
      2. From the gRNA output page on http://grna.ctegd.uga.edu/, select a 20 nt gRNA protospacer sequence with an NGG PAM downstream of the translation stop codon (e.g., STOP-N…N<100GATCGACTTAGTAACGCATCCGG) (Figure 2E). Optional: Add a ‘G’ to the 5’ end of the protospacer sequence if it does not begin with one to enhance sgRNA transcription (Doench et al., 2014).
    3. Mutagenesis of pSAG1::Cas9-U6::sgUPRT (Addgene #54467) (Shen et al., 2014)
      1. The plasmid pSAG1::Cas9-U6::sgUPRT transiently expresses SpCas9-HA-GFP nuclease and a single guide RNA (sgRNA) that binds Cas9 and directs it to perform a double stranded break in the TgUPRT gene. The gene target specificity of the Cas9/sgRNA ribozyme is based on a 20 nt protospacer sequence at the 5’-most end of the sgRNA. To switch the Cas9 target from TgUPRT to a different gene, the 20 nt protospacer sequence will need to be changed to the one designed in Step A2 Protospacer Selection. Use Q5 Site-Directed Mutagenesis (New England Biolabs) to mutate pSAG1::Cas9-U6::sgUPRT, changing the 20 nt TgUPRT protospacer to the 20 nt protospacer selected for the gene of interest (Figure 2E). A standard 25 µl reaction consists of:
        1 µl (1 ng) pSAG1::Cas9-U6::sgUPRT
        1.25 µl (10 µM) reverse primer: (5’-AACTTGACATCCCCATTTAC)
        1.25 µl (10 µM) forward primer:
        (5’-(G)NNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGC)
        9 µl deionized H2O
        12.5 µl 2x Q5 Hot-Start PCR mix
      2. Q5 Mutagenesis thermal cycling
        Step 1. Denature at 98 °C for 30 sec
        Step 2. Denature at 98 °C 10 sec
        Step 3. Anneal at 55 °C 30 sec
        Step 4. Extend at 72 °C for 5 min
        Step 5. Repeat Steps 2-4 24 times
        Step 6. Extend at 72 °C for 2 min
        Step 7. Hold at 4 °C until further use.
      3. Resolve 1 µl of PCR reaction on a 0.8% agarose gel (TAE or TBE) with a 1 kb DNA ladder to check for amplification. The expected amplicon size is 9.68 kb.
      4. Kinase, Ligase, Digest (KLD) reaction (included in the Q5 Site-Directed Mutagenesis Kit)
        1 µl PCR product
        3 µl deionized H2O
        5 µl 2x KLD buffer
        1 µl 10x KLD enzyme mix
        Mix and incubate at room temperature for 5 min.
      5. Transform NEB5α chemically-competent E. coli with 1 µl of the KLD reaction. Incubate on ice for 30 min. Heat chemically-transformed cells for 30 sec at 42 °C. Add 950 µl SOC medium (New England Biolabs) to transformed cells and incubate at 37 °C with mixing for 1 h. Spread 100 µl of transformed E. coli onto LB agar plates with 100 µg/ml ampicillin. Incubate plates at 37 °C overnight. The next day, pick 2-3 colonies from each LB agar plate to grow up in 5 ml LB broth + 100 µg/ml ampicillin overnight cultures for plasmid extraction.
      6. Pellet overnight bacterial cultures at 3,000 x g with a benchtop centrifuge for 10 min.
      7. Extract plasmid DNA from bacterial pellets using a plasmid extraction kit (Macherey-Nagel). Pre-warm sterile-filtered Buffer EB to 70 °C. Elute DNA into a sterile 1.7 ml microfuge tube with 50 µl pre-warmed sterile-filtered Buffer EB.
      8. Measure DNA concentration and purity by spectrophotometry (absorbance at 260 nm/280 nm, NanoDrop (Thermo Fisher)). The expected yield is approximately 500-1,000 ng/µl.
      9. Confirm mutated protospacer sequence of pSAG1::Cas9-U6::sg”GOI” by Sanger sequencing using the M13 reverse universal primer (5’-ACAGGAAACAGCTATGAC) (Genewiz).


        Figure 2. Generation of constructs for C-terminal mAID tagging. A-E. Generating gene-specific Cas9/sgRNA plasmids. The gene page of TgPKG (TGGT1_311360; example gene for C-terminal tagging) from ToxoDB.org. The red arrow points to the download gene link for acquiring the TgPKG genomic sequence needed. B. The ‘Download Gene’ page for TgPKG from ToxoDB.org. The red asterisks highlight fields that need to be entered as shown. The red arrow points to the get sequences link. C. The FASTA genomic sequence of TgPKG (-80 to +200 from the translation stop codon) from ToxoDB.org needed for gRNA and donor homology design for C-terminal tagging. D. Guide RNA design tool (cropped to fit) from EuPaGDT (http://grna.ctegd.uga.edu/). The red asterisks indicate the basic fields that need to be completed. The red arrow points to the ‘Get guide RNA’ link. E. Output of gRNA protospacers for TgPKG genomic sequence (-80 to +200 from the translation stop codon) on each strand from EuPaGDT (upper schematic). Selected gRNA protospacer and protospacer adjacent motif (PAM) sequence with quality scores from EuPaGDT (table). Swapping the TgUPRT protospacer with the designed TgPKG protospacer in pSAG1::Cas9-U6::sgUPRT by site-directed mutagenesis (lower schematic). F. Generation of a TgPKG-specific C-terminal mAID-3HA, HXGPRT tagging cassette. The FASTA genomic sequence of TgPKG (-80 to +200 from the translation stop codon) from ToxoDB.org with the indicated highlighted features. PCR amplification of mAID-3HA, HXGPRT tagging cassette from pYFP-mAID-3HA, Floxed HXGPRT with 5’ and 3’ regions of donor homology (40 bp) to TgPKG (flanking the Cas9 break-site) added to the forward and reverse primers, respectively (asterisks).

  2. Generation of a gene-specific AID tagging cassette
    1. Microhomology flank selection and primer design
      1. The 5’ microhomology flank is the last 40 nt of the gene of interest that immediately precedes the translation stop codon. The 3’ microhomology flank will be the first 40 nt that immediately follows the selected protospacer/PAM site. These flanks will be appended to the (m)AID tagging cassette by PCR using ~60 nucleotide DNA oligo primers (Figure 2F).
      2. Order tagging primers. The tagging forward primer will be the 40 nt 5’ homology flank + GCTAGCAAGGGCTCGGGC. The tagging reverse primer will be the 40 nt 3’ homology flank (reverse complement) + ATAGGGCGAATTGGAGCTCC. We purchase 25 nmole DNA oligo primers with ‘standard desalting’ from Integrated DNA Technologies (www.idtdna.com).
    2. PCR amplification and clean-up of an AID tagging amplicon
      1. Q5 PCR set up. Set up the Q5 PCR reaction using pYFP-mAID-3HA, Floxed HXGPRT (Addgene #87259) (Brown et al., 2017) or pYFP-AID-3HA, Floxed HXGPRT (Addgene #87260) (Long et al., 2017b) plasmids as the template (Figure 2F). A standard 500 µl reaction consists of:
        100 µl 5x Q5 Buffer
        10 µl dNTPs
        5 µl (50 µM) tagging forward primer (Figure 2F)
        5 µl (50 µM) tagging reverse primer (Figure 2F)
        5 µl (5 ng) plasmid template
        5 μl Q5 DNA polymerase
        370 µl deionized H2O
        Split the PCR mix into 5 PCR tubes, 100 µl each
      2. Q5 PCR thermal cycling.
        Step 1. Denature at 98 °C for 30 sec
        Step 2. Denature at 98 °C for 10 sec
        Step 3. Anneal at 60 °C for 30 sec
        Step 4. Extend at 72 °C for 2.5 min
        Step 5. Repeat Steps 2-4 29 times
        Step 6. Extend at 72 °C for 2.5 min
        Step 7. Hold at 4 °C until further use
      3. Checking PCR quality. Resolve 1 µl of PCR reactions on 0.8% agarose gel (TAE or TBE) with a 1 kb DNA ladder to check for amplification. The tagging amplicon will consist of a 5’ homology flank-linker-(m)AID-3HA, Floxed HXGPRT-3’ homology flank (expected amplicon sizes: 2.9 kb for mAID, 3.4 kb for AID).
      4. PCR cleanup with a PCR Cleanup Kit (Macherey-Nagel). Pool the 5 PCR reactions and add 1,000 µl Buffer NTI. Load 750 µl mix into DNA column. Centrifuge at 11,000 x g with a microfuge for 1 min. Discard flow through. Load remaining 750 µl mix into the column. Centrifuge at 11,000 x g for 1 min. Discard flow through. Wash column with 700 µl Buffer A3. Centrifuge at 11,000 x g for 1 min. Discard flow through. Repeat wash step and discard flow through. Centrifuge additional 3 min at 11,000 x g. Pre-warm sterile-filtered Buffer EB to 70 °C. Elute DNA into a sterile 1.7 ml microfuge tube with 30 µl pre-warmed sterile-filtered Buffer EB.
      5. Measure DNA concentration and purity by spectrophotometry (Absorbance at 260 nm/280 nm, NanoDrop (Thermo Fisher)). Aim for having at least 10 µg tagging cassette for parasite transfection. Repeat PCR and cleanup steps if more DNA is needed.

  3. Cell culture
    1. Human foreskin fibroblasts (HFF) culture
      1. Starter cultures. Grow starter HFF cells in 35 ml D10 medium (see Recipes) in sterile T-175 flasks in a 37 °C 5% CO2 incubator.
      2. HFF passage. Once confluent, remove D10 medium and wash with 35 ml sterile 1x PBS and discard wash. Add 5 ml trypsin, replace cap, and incubate cells at 37 °C for 1-5 min until cells detach from one another (as detected using 10x magnification). Firmly tap the side of the flask a few times forcefully to detach cells from the plastic. Add 175 ml fresh D10 medium to trypsinized cells and mix to generate a 1:5 cell suspension. Passage 35 ml into new T-175. Passage 5 ml into T-25s to make HFF feeder flasks to support Toxoplasma gondii culture. Passage 200 µl per well of 96-well plates and 1 ml per well of 24-well plates for parasite cloning following transfection.
    2. Parasite culture
      1. Parasite information. The T. gondii line RH TIR1-3FLAG (genotype: RHΔhxgprtΔku80; TUB1:TIR1-3FLAG, SAG1:CAT) is needed as it provides three key elements for the AID system: Δku80 restricts non-homologous end-joining, reducing random integration of the AID tagging cassette; Δhxgprt provides the proper genetic background for positive selection of the AID tagging cassette based on HXGPRT-mediated resistance to Mycophenolic acid; TIR1-3FLAG is the plant auxin receptor required for in auxin induced SCFTIR1-targeting of AID-tagged proteins for proteasomal degradation.
      2. Parasite passage. Inoculate confluent HFF T-25 feeder flasks with RH TIR1-3FLAG parasites using 350 µl of freshly egressed parasites every two days or immediately following natural egress as needed. Culture parasites in 37 °C 5% CO2 in D10 medium.

  4. (m)AID tagging using CRISPR genome editing (e.g., TgPKG-mAID-3HA; Figure 3)
    1. Parasite transfection
      1. Culture parasites for transfection. Infect confluent host cells grown in T-25 flasks with sufficient RH TIR1-3FLAG parasites (95 µl of freshly egressed parasites from T-25) to achieve around 75% host cell lysis 2 days post-infection. Scrape the monolayers and pass the cell suspension through a 22 G blunt-end needle 3-5 times to disrupt the host cells and release all parasites. Avoid needle sticks and carefully dispose of used needles into a biohazard sharps container. Pre-wet 3.0 μm polycarbonate membranes by passing 5 ml HHE through the filter with a 10 ml syringe into a waste container. Pass the syringe-lysed parasites through the filter with a 10 ml syringe into a 50 ml polystyrene conical vial. Pass an additional 5 ml HHE through the filter into the 50 ml conical to collect additional parasites from the filter. Centrifuge the filtered culture at 400 x g with a benchtop centrifuge for 10 min at 18 °C, resuspend the pellets in 10 ml HHE, count the cell density with a hemacytometer, and centrifuge again under the same conditions. Subsequently, resuspend the parasite cell pellet in Cytomix buffer (see Recipes) to achieve a parasite density of 4 x 107 ml-1.
      2. Prepare the transfection mix in an electroporation cuvette
        250 µl parasites in Cytomix buffer
        10 µg GOI-specific CRISPR/Cas9 plasmid (Figures 2A-2E)
        10 µg GOI-specific AID-tagging PCR amplicon (Figure 2F)
        3 µl ATP (0.2 M, 100x)
        3 µl glutathione (0.5 M, 100x)
        Adjust to 300 µl with Cytomix buffer.
      3. Electroporation. Electroporate parasites according to electroporator manufacturer’s instructions. We use the BTX ECM-830 electroporator with 4 mm gap cuvettes, with the following parameters:
        1,700 V
        176 μsec of pulse length
        2 pulses
        100 msec interval between pulses
        Immediately after electroporation, transfer the parasites into fresh T-25 flasks with confluent host cells and grow them at 37 °C, 5% CO2.
    2. Drug selection and parasite cloning
      1. Drug selection. At 24 h after electroporation and recovery, begin drug selection for the (m)AID-3HA, Floxed HXGPRT construct using mycophenolic acid (25 µg/ml) supplemented with xanthine (50 µg/ml). Repeat passages until the drug-resistant pool becomes stable (usually 2-3 passages until the culture stabilizes (i.e., lyses the monolayer every two days with a 350 µl inoculum of freshly egressed parasites).
      2. Parasite cloning by limiting dilution. Following natural egress of the drug-resistant population, purify and count the parasites as described above. Dilute the purified parasites with D10 medium (without drug) to achieve 3 parasites/150 μl. Subsequently, add 150 μl/well of this diluted culture into 96-well plates with confluent host cells and let the parasites grow at 37 °C, 5% CO2 for 7 days without movement to allow plaques (macroscopic focal destruction of the host cell monolayer by lytic parasite replication) to develop.
    3. Screening clones for genetic recombination
      1. Identifying clones. After plaque development, visually check each well of the 96-well plates under an inverted-phase contrast microscope (such as a Nikon TS100) and look for wells that contain only one plaque (Refer to Ufermann et al., 2017 for high resolution examples of plaque morphology). Transfer the parasites of positive wells into 24-well plates containing confluent host cell monolayers and grow them until they naturally egress.
      2. Making lysates for diagnostic PCR. Inoculate a small aliquot (e.g., 10 μl) from each well into a new 24-well plate containing a confluent monolayer of host cells to continue culturing the parasites. The rest of the lysed out culture will be used for diagnostic PCR. Add 1 ml egressed parasite clones into 1.7 ml microfuge tubes. For a negative control, add 1 ml of egressed wild-type parasites (the parental line) to a separate 1.7 ml microfuge tube. Pellet the parasite suspensions at 400 x g with a microfuge for 10 min at 18 °C and remove supernatant from parasite pellet. To each pellet, add 100 μl Proteinase K (PK) (0.2 mg/ml) in 1x PBS and mix. Heat at 37 °C for 10 min, 50 °C 10 min, and 98 °C for 10 min. Freeze samples or chill on ice and proceed with diagnostic PCR.
      3. Diagnostic PCR set up. Two simple PCRs should be performed to determine the tagging success. PCR1 should exclusively amplify the unmodified gene locus, while PCR2 should exclusively amplify the tagged gene locus (Figure 3A). Each 25 μl diagnostic PCR reaction consists of:
        2.5 μl 10x Taq buffer
        0.5 μl dNTPs
        0.25 μl (50 μM) forward primer (same for PCR1 and PCR2)
        0.25 μl (50 μM) reverse primer (unique for PCR1 and PCR2)
        1 μl PK-treated parasite lysate
        0.25 μl Taq DNA polymerase (New England Biolabs)
        20.25 μl deionized H2O
        The forward primer for PCR1 and PCR2 should prime ~350 bp upstream (5’ side) of the translation stop codon. The PCR1 reverse primer should prime in the 3’ UTR, ~300 bp downstream of the translation stop codon. The PCR2 reverse primer should prime in the mAID or AID sequence ~+50 from start of linker.
      4. Diagnostic PCR thermal cycling
        Step 1. Denature at 98 °C for 30 sec
        Step 2. Denature at 98 °C 10 sec
        Step 3. Anneal at 60 °C 30 sec
        Step 4. Extend at 68 °C for 1 min
        Step 5. Repeat Steps 2-4 29 times
        Step 6. Extend at 68 °C for 1 min
        Step 7. Hold at 4 °C until further use
      5. Resolving diagnostic PCR amplicons. Resolve 10 µl of PCR reactions on 1% agarose gel (TAE or TBE) with a 1 kb DNA ladder to check for positive amplification (Figure 3C). PCR1 should yield an amplicon of ~650 bp for the wild-type, unmodified locus. PCR2 should yield an amplicon of ~400 bp for successfully tagged clones. PCR positive clones can be further examined by other means such as immunofluorescence microscopy (Figure 3D) or Western blotting (Figure 3E) for the (m)AID-3HA epitope tag using anti-HA antibodies.
      6. Subcloning tagged clones. Once positive clones are identified and confirmed, they should be transferred from 24-well plates into T-25 flasks and cryopreserved for future use.


        Figure 3. Generation and regulation of (m)AID protein fusions. A. CRISPR-mediated C-terminal mAID tagging in RH TIR1-3FLAG line. The example shown here is the gene encoding protein kinase G (TgPKG, reproduced with permission from [Brown et al., 2017]). Note the locations of diagnostic PCRs 1 and 2. B. Schematic of TgPKG-mAID-3HA expression showing the two PKG-mAID isoforms that are generated from alternative translation initiation sites on the transcript. Addition of IAA promotes the simultaneous degradation of both PKG-mAID isoforms. C-D. Data reproduced with modification for space limits from (Brown et al., 2017). C. Diagnostic PCRs 1 and 2 from genomic DNA samples showing successful integration of the mAID tagging construct into TgPKG (see A for PCR positions). WT (wild type), TIR1-3FLAG parent; Tag, PKGI,II-mAID-3HA parasites. D. Immunofluorescence of PKG-mAID-3HA isoforms I and II stained with anti-HA mAb and detected with Alexa Fluor 488-conjugated secondary antibody. Scale bar = 5 µm. E. Western blot assay of lysed PKGI,II-mAID-3HA parasites probed with antibodies recognizing HA (green) and aldolase (red). Parasites were treated with 500 µM IAA or the vehicle (EtOH) for 4 h prior to lysis.

  5. Depletion of AID-tagged proteins
    1. Parasite culture. Infect HFF cultures with RH TIR1-3FLAG and RH POI-(m)AID-3HA parasites. The type (e.g., dish, well plate, flask) and number of HFF monolayers to infect will depend on the experiment but at least two cultures are needed per parasite line for auxin and vehicle treatment.
    2. Auxin treatment. Use auxin at 1:1,000 to deplete (m)AID-tagged proteins at a final concentration of 500 µM. Mock treatment consists of an equivalent volume of 100% EtOH at a final concentration of 0.0789% (w/v). Incubate at 37 °C. Incubation times needed to achieve complete protein knockdown will vary based on protein localization, stability, and abundance. As little as 15 min has been used to achieve complete knockdown of soluble cytosolic (m)AID-fusions such as YFP (Brown et al., 2017; Long et al., 2017b). Following knockdown, parasites can be examined for phenotypes directly, harvested for follow-up assays, or lysed for analysis of (m)AID-tagged protein levels.
    3. Detection of AID-tagged protein levels. The 3HA portion of the (m)AID construct can be used for protein detection by immunofluorescence microscopy (Figure 3D) or Western blotting (Figure 3E) using anti-HA antibodies (Figure 2).

Data analysis

Quantification of Western blot data of (m)AID fusion knockdowns performed in triplicate can be performed using ImageJ densitometry software. We recommend viewing an online resource created by Dr. Luke Miller of San Jose State University (http://lukemiller.org/index.php/2010/11/analyzing-gels-and-western-blots-with-image-j/) for detailed guidelines for ImageJ densitometry.

  1. In ImageJ, open the grayscale TIF image of the (m)AID-3HA anti-HA Western blot. File -> open ->.tif
  2. With the rectangle tool, draw a vertical rectangle around the protein band in the first lane. The rectangle must be taller than it is wide.
  3. Press Ctrl + 1 to select the first lane. A ‘1’ will appear in the rectangle.
  4. Left click the rectangle and drag it to the next lane.
  5. Press Ctrl + 2 to select the second lane. A ‘2’ will appear in the second rectangle.
  6. Left click the newest rectangle and move to the third lane. Repeat Steps 5 and 6 until every lane has a rectangle.
  7. Press Ctrl + 3 to plot the data in a histogram. A new window will pop up.
  8. With a line drawing tool, draw a line under each peak that connects the background line on the left to the background line on the right.
  9. After all background lines are drawn, use the ‘magic wand’ tool to click inside of each peak, starting at the top (lane 1), and working downward.
  10. The density values for each peak (band) will be found in a new pop-up table.
  11. Repeat Steps 1-11 for the loading control blot.
  12. Normalize for total protein (AID-3HA density/loading control density) for each sample then transform this ratio as a percentage of the untreated control (normalized sample value/normalized control ratio x 100).
  13. The data from triplicate experiments can be averaged and analyzed for statistical significance using GraphPad Prism software according to manufacturer’s instructions.

Notes

The T. gondii line RH TIR1-3FLAG (genotype: RHΔhxgprtΔku80; TUB1:TIR1-3FLAG, SAG1:CAT) (Brown et al., 2017; Long et al., 2017b) has been submitted to the ATCC for distribution. To apply the auxin degron system to other T. gondii strains, the plasmid pTUB1:OsTIR1-3FLAG, SAG1:CAT (Addgene #87258) is available for transfection. Please visit http://www.addgene.org/David_Sibley/ for other AID-related plasmids.

Recipes

  1. 10x Tris-Acetate-EDTA (TAE) buffer
    48.4 g Tris base
    11.42 ml glacial acetic acid
    20 ml 0.5 M EDTA pH 8.0
    Q.S. to 1 L with deionized H2O
    Store at room temperature
  2. 10x Tris-Borate-EDTA (TBE) buffer
    108 g Tris base
    55 g boric acid
    40 ml 0.5 M EDTA pH 8.0
    Q.S. to 1 L with deionized H2O
    Store at room temperature
  3. 10x SDS-PAGE running buffer
    30 g Tris base
    144 g glycine
    10 g SDS
    Q.S. to 1 L with deionized H2O
    Store at room temperature
  4. 10x protein transfer buffer
    30.3 g Tris base
    144 g glycine
    Q.S. to 1 L with deionized H2O
  5. 1x protein transfer buffer
    700 ml deionized H2O
    200 ml methanol
    100 ml 10x protein transfer buffer
  6. 10x phosphate buffered saline (PBS)
    2 g KCl
    80 g NaCl
    14.4 g Na2HPO4
    2 g KH2PO4
    Q.S. to 1 L with deionized H2O
    Store at room temperature
  7. Phosphate buffered saline + Tween-20 (PBST)
    899 ml deionized H2O
    100 ml 10x PBS
    1 ml Tween-20
    Store at room temperature
  8. PCR lysis buffer
    0.2 mg/ml Proteinase K in PBS
  9. D10 medium
    1 packet of DMEM powder
    3.7 g sodium bicarbonate
    2.38 g HEPES
    10 ml of 200 mM L-glutamine
    1 ml of 10 mg/ml gentamicin
    100 ml Characterized fetal bovine serum
    Q.S. to 1 L with deionized H2O
    Filter sterilize with 1 L Stericup Filter and store at 4 °C
  10. Hank’s balanced salt solution with HEPES and EGTA (HHE)
    Hanks’ balanced salt solution containing 10 mM HEPES and 1 mM EGTA
  11. 0.1 M KPO4 buffer pH 7.6 (for Cytomix buffer)
    17.4 g K2HPO4
    13.6 g KH2PO4
    Q.S. to 1 L with deionized H2O
  12. Cytomix electroporation buffer pH 7.6 (Soldati and Boothroyd, 1993)
    100 ml 0.1 M KPO4 pH 7.6
    8.95 g KCl
    22.1 mg CaCl2
    1.02 g MgCl2
    5.96 g HEPES
    744 mg EDTA
    Q.S. to 1 L with deionized H2O
    Sterile filter and store at 4 °C
  13. 500 mM 3-indoleacetic acid (IAA/auxin) (1,000x stock)
    87.59 mg 3-indoleacetic acid into 1 ml 100% EtOH
    Store at -20 °C, protect from light

Acknowledgments

We would like to thank Dr. Nisha Philip and Dr. Andy Waters for helpful discussions and sharing plasmids for the development of the AID protocol in T. gondii. We have no conflicts of interest to declare.

References

  1. Brown, K. M., Long, S. and Sibley, L. D. (2017). Plasma membrane association by N-acylation governs PKG function in Toxoplasma gondii. MBio 8(3).
  2. Doench, J. G., Hartenian, E., Graham, D. B., Tothova, Z., Hegde, M., Smith, I., Sullender, M., Ebert, B. L., Xavier, R. J. and Root D. E. (2014). Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol 32(12):1263-7.
  3. Kreidenweiss, A., Hopkins, A.V., and Mordmuller, B. (2013). 2A and the auxin-based degron system facilitate control of protein levels in Plasmodium falciparum. PLoS One 8(11): e78661.
  4. Long, S., Brown, K. M., Drewry, L. L., Anthony, B., Phan, I. Q. H. and Sibley, L. D. (2017a). Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii. PLoS Pathog 13(5): e1006379.
  5. Long, S., Anthony, B., Drewry, L., and Sibley, L. D. (2017b). A conserved ankyrin repeat-containing protein regulates conoid stability, motility and cell invasion in Toxoplasma gondii. Nat Commun 8(1): 2236.
  6. Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T. and Kanemaki, M. (2009). An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods 6(12): 917-922.
  7. Philip, N. and Waters, A. P. (2015). Conditional degradation of Plasmodium calcineurin reveals functions in parasite colonization of both host and vector. Cell Host Microbe 18(1): 122-31.
  8. Shen, B., Brown, K., Long, S. and Sibley, L. D. (2017). Development of CRISPR/Cas9 for efficient genome editing in Toxoplasma gondii. Methods Mol Biol 1498: 79-103.
  9. Shen, B., Brown, K. M., Lee, T. D. and Sibley, L. D. (2014). Efficient gene disruption in diverse strains of Toxoplasma gondii using CRISPR/Cas9. MBio 5(3): e01114-01114.
  10. Sidik, S. M., Hackett, C. G., Tran, F., Westwood, N. J. and Lourido, S. (2014). Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9. PLoS One 9(6): e100450.
  11. Soldati, D. and Boothroyd, J. C. (1993). Transient transfection and expression in the obligate intracellular parasite Toxoplasma gondii. Science 260(5106): 349-352.
  12. Starnes, G. L., Coincon, M., Sygusch, J. and Sibley, L. D. (2009). Aldolase is essential for energy production and bridging adhesin-actin cytoskeletal interactions during parasite invasion of host cells. Cell Host Microbe 5:353-364.
  13. Teale, W. D., Paponov, I. A. and Palme, K. (2006). Auxin in action: signalling, transport and the control of plant growth and development. Nat Rev Mol Cell Biol 7(11): 847-859.
  14. Ufermann, C-M., Muller, F., Frohnecke, N., Laue, M. and Seeber, F. (2017). Toxoplasma gondii plaque assays revisited: Improvements for ultrastructural and quantitative evaluation of lytic parasite growth. Exp Parasitol 180:19-26.

简介

弓形虫是原生动物寄生虫称为Apicomplexa致命门的一员。 作为一个复杂的模型,关于T的信息有很多。 gondii的8,000多种蛋白质编码基因,包括序列变异,表达和对寄生虫适应的相对贡献。 然而,需要新的工具来功能性地调查数百个推定的必需蛋白质编码基因。 因此,我们最近实施了生长素诱导降解(AID)系统来研究T中的基本蛋白质。弓形虫。 在这里,我们提供了一个检查蛋白质功能的一步一步的协议。 在组织培养环境中使用AID系统。

【背景】生长素是一类通过靶向某些蛋白质在植物中进行蛋白酶体降解而发出信号的植物激素(Teale等人,2006)。 Kohei Nishimura等人具有将该植物特异性信号传导系统的组分转移到其他真核生物中用于有兴趣的蛋白质(POI)的条件调节,创建生长素诱导降解(AID)系统的聪明想法(Nishimura等人,2009)。这个系统已经被成功地用于几种真核生物,包括疟原虫疟原虫(Kreidenweiss et al。,2013; Philip和Waters,2015)。只需要两个转基因成分来实现这个系统,称为转运抑制剂反应1(TIR1)的植物生长素受体和用AID标记的POI。用生长素(例如,3-吲哚乙酸/ IAA)处理活化SCF TIR1泛素连接酶复合物,其专一地靶向AID标记的蛋白质以获得泛素依赖性蛋白酶体降解(图1)。我们最近设计了一个RH hxgprt ku80 T的行。 (RH TIR1-3FLAG)(Brown等人,2017; Long等人, )稳定表达TIR1。 >,2017)。在此背景下,我们能够使用CRISPR / Cas9基因组编辑(Shen等人,2014; Sidik等人,2014; Shen&amp; em; et al。 ,2017)来标记必要的 T。 (AID-3HA)或迷你AID(mAID)-3HA感兴趣的弓形虫基因,用生长素调节它们的表达,并鉴定与其丧失相关的表型(Brown等人,2017; Long&lt; em&gt;等人,2017a和2017b)。

使用这个系统在 T的条件击倒有几个好处。弓形虫。首先,POI-AID融合体从其内源启动子表达,维持正常的表达时间和水平。其次,生长素对寄生虫和宿主细胞培养物无毒性,1 mM,但功能低至〜50μM。第三,生长素仅在需要降解时添加,并且以每克低于5美元的价格商购。最后也是最重要的是,POI-AID融合体在生长素处理后15分钟内完全降解。由于这些原因,我们不得不在这个详细的协议中详细说明我们公布的方法,以便在其他尖端的实验室中建立这个系统。

“”src
A.植物生长素受体TIR1在没有生长素的情况下处于无活性的“Apo”状态,允许感兴趣的蛋白(POI) - AID-3HA融合表达和功能正常。 B.生长素结合的TIR1组装成活跃的Skp-Cullen-F盒(SCF TIR1)泛素连接酶复合物,其识别和多遍在蛋白化AID。 C.多聚遍在蛋白修饰靶向用于蛋白酶体降解的POI-AID-3HA。

关键字:生长素, 降解因子, AID, 条件性敲减, 蛋白质调节, 寄生虫, 刚地弓形虫, CRISPR

材料和试剂

  1. 微量离心管(1.7毫升)
  2. PCR管(0.2毫升)
  3. T-25和T-175培养瓶(Corning,目录号:430639,431080)
  4. 96孔组织培养板(TPP,目录号:92696)
  5. 24孔板(TPP,目录号:92024)
  6. 22 G钝针(CML Supply,目录号:901-22-100M)
  7. 3.0μm孔径47 mm滤膜(GE Healthcare,Whatman,目录号:111112)
  8. 10毫升注射器(BD,目录号:309695)
  9. 50毫升聚苯乙烯锥形小瓶(Fisher Scientific,目录号:05-539-10)
  10. 1升Stericup过滤器(默克,目录号:SCVPU11RE)
  11. 13毫米细胞刮刀(TPP,目录号:99002)
  12. 47毫米聚碳酸酯注射器过滤器支架(GE Healthcare,Whatman,目录号:420400)
  13. 用于Western印迹湿法转移的滤纸(GE Healthcare,Whatman,目录号:3030-917)
  14. 硝化纤维素膜(GE Healthcare,Amersham,目录号:10600003)
  15. 培养皿(Sigma-Aldrich,目录号:P5606)
  16. Gilson移液器吸头(Gilson,产品目录号:F171101,F171301,F171501)
  17. 无菌血清移液器(5毫升,10毫升,25毫升)
  18. 电穿孔比色杯4毫米(BTX,目录号:45-0126)
  19. pSAG1 :: Cas9-U6 :: sgUPRT质粒(Addgene,目录号:54467)(Shen等人,2014)
  20. pYFP-AID-3HA,Floxed HXGPRT质粒(Addgene,目录号:87260)(Long等人,2017)。
  21. pYFP-mAID-3HA,Floxed HXGPRT质粒(Addgene,目录号:87259)(Brown等人,2017)。
  22. 吨。弓形虫RH TIR1-3FLAG(基因型:RHΔ hxgprt Δku80 ; TUB1 :TIR1-3FLAG,SAG1 : )(Brown et。,2017; Long et al。,2017)
  23. NEB5α化学胜任的E。用SOC培养基(New England Biolabs,目录号:C2987I)检测大肠杆菌
  24. 人包皮成纤维细胞(HFF)(ATCC,目录号:SCRC-1041)
  25. Q5定点突变试剂盒,具有化学能力 E。 (New England Biolabs,目录号:E0554S)
  26. 用于重编程pSAG1 :: Cas9-U6 :: sgUPRT(IDT,25nmole,标准脱盐)的诱变引物
  27. 2x SDS-PAGE样品缓冲液(Sigma-Aldrich,目录号:S3401)
  28. 1 kb的DNA梯(新英格兰生物实验室,目录号:N3232)
  29. 琼脂糖(Fisher Scientific,目录号:BP160)
  30. SDS-PAGE 4-15%梯度Tris-甘氨酸聚丙烯酰胺凝胶(Bio-Rad Laboratories,目录号:4561086)
  31. 6X凝胶加载染料(新英格兰生物实验室,目录号:B7025)
  32. LB肉汤(BD,目录号:244610)
  33. 氨苄青霉素(Sigma-Aldrich,目录号:A9518)
  34. 质粒小量制备试剂盒(Macherey-Nagel,目录号:740588)
  35. M13反向通用引物(5'-ACAGGAAACAGCTATGAC)(Genewiz)
  36. Q5 DNA聚合酶(New England Biolabs,目录编号:M0491)
  37. dNTP各10mM(New England Biolabs,目录号:N0447)
  38. 用于扩增(m)AID-3HA的兴趣标签引物基因,具有短同源性侧翼(IDT,25nmole,标准脱盐)的Floxed HXGPRT标签盒
  39. 基因目标诊断标记引物(IDT,25 nmole)
  40. 琼脂糖凝胶和PCR清除试剂盒(Macherey-Nagel,目录号:740609)
  41. 胰蛋白酶-EDTA(Sigma-Aldrich,目录号:T3924)
  42. ATP(Sigma-Aldrich,目录号:A6419)
  43. 谷胱甘肽(Sigma-Aldrich,目录号:G6013)
  44. 霉酚酸(Sigma-Aldrich,目录号:M3536)
  45. 黄嘌呤(Sigma-Aldrich,目录号:X4002)
  46. 蛋白酶K(Sigma-Aldrich,目录号:P2308)
  47. Taq聚合酶(New England Biolabs,目录号:M0273)
  48. 乙醇(EtOH)(Pharmco-AAPER,目录号:11100020)
  49. GelRed核酸染色剂(Biotium,目录号:41001)
  50. Licor抗小鼠800CW二抗(LI-COR,目录号:925-32210)
  51. Licor抗兔680RD二抗(LI-COR,目录号:925-68071)
  52. 小鼠抗HA单克隆抗体(BioLegend,目录号:901501)
  53. 不含脂肪的奶粉(雀巢康乃馨)
  54. 兔抗醛缩酶(弓形虫)(Starnes等人,2009)或其他T细胞。弓形虫加载对照抗体
  55. Tris碱(Sigma-Aldrich,目录号:T6066)
  56. 冰醋酸(Fisher Scientific,目录号:A38-500)
  57. 0.5M EDTA pH 8.0(Merck,目录号:324504)
  58. 硼酸(Sigma-Aldrich,目录号:B6768)
  59. 甘氨酸(Sigma-Aldrich,目录号:G7128)
    注:此产品已停产。
  60. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L5750)
  61. 甲醇(Fisher Scientific,目录号:A412P)
  62. 氯化钾(KCl)(Sigma-Aldrich,目录号:P5405)
  63. 氯化钠(NaCl)(Fisher Scientific,目录号:S271)
  64. 磷酸二氢钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S3264)
  65. 磷酸二氢钾(KH 2 HPO 4)(Sigma-Aldrich,目录号:P8281)
  66. 吐温-20(Sigma-Aldrich,目录号:P2287)
  67. 达尔伯克改良伊格尔培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:12100046)
  68. 碳酸氢钠(Sigma-Aldrich,目录号:S5761)
  69. 200mM L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030149)
  70. 10mg / ml庆大霉素(Thermo Fisher Scientific,Gibco TM,产品目录号:15710072)
  71. 表征的胎牛血清(FBS)(GE Healthcare,目录号:SH30071.01HI)
  72. Hanks平衡盐溶液(Sigma-Aldrich,目录号:H9269)
  73. HEPES(Sigma-Aldrich,目录号:H3375)
  74. EGTA(Merck,目录号:324626)
  75. EDTA(Merck,目录号:324504)
  76. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  77. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M4880)
  78. 氯化钙二水合物(CaCl 2•2H 2 O)(Sigma-Aldrich,目录号:C5080)
  79. 3-吲哚乙酸(IAA /生长素)(Sigma-Aldrich,目录号:I2886)
  80. 琼脂(Fisher Scientific,目录号:BP1423)
  81. 10X Tris-Acetate-EDTA(TAE)缓冲液(见食谱)
  82. 10倍Tris-Borate-EDTA(TBE)缓冲液(见食谱)
  83. 10倍SDS-PAGE运行缓冲液(见食谱)
  84. 10倍蛋白转移缓冲液(见食谱)
  85. 1x蛋白转移缓冲液(见食谱)
  86. 10倍磷酸盐缓冲盐水(PBS)(见食谱)
  87. 磷酸盐缓冲盐水+吐温-20(PBST)(见食谱)
  88. PCR裂解缓冲液(见食谱)
  89. D10中(见食谱)
  90. Hank平衡盐溶液与HEPES和EGTA(HHE)(见食谱)
  91. 0.1M KPO 4缓冲液pH 7.6(用于Cytomix缓冲液)(参见食谱)
  92. Cytomix电穿孔缓冲液pH 7.6(Soldati和Boothroyd,1993)(见食谱)
  93. 500毫摩尔3-吲哚乙酸(IAA /生长素)(1,000×股票)(见食谱)

设备

  1. 高压灭菌器(Steris,型号:SG-120)
  2. 台式离心机与15毫升和50毫升锥形小瓶持有人(Eppendorf,型号:5810 R)
  3. 生物安全柜(贝克,型号:SterilGuard®II)
  4. CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma TM 310)。
  5. 不含洗涤剂的派热克斯玻璃器皿和搅拌棒用于培养基制备
  6. Electroporator(BTX,型号:ECM 830)
  7. 凝胶文件系统(Bio-Rad Laboratories,型号:Gel Doc TM XR +)
  8. 孵化轨道摇床(VWR,型号:3500I型)
  9. 倒置相差显微镜(Nikon Instruments,型号:Eclipse TS100)
  10. 血细胞计数器(Sigma-Aldrich,型号:Bright-Line TM)
  11. Licor Odyssey成像系统(LI-COR,型号:Odyssey®CLx)
  12. 微量离心机(Eppendorf,型号:5417 R)
  13. 微量分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM TM One)
  14. 微波炉(VWR,目录号:75856-526)
    制造商:Argos Technologies,目录号:111092。
  15. 移液助剂(Thermo Fisher Scientific,Thermo Scientific TM,型号:S1)
  16. 移液器(Pipetman,Gilson,型号:P2,P20,P200,P1000)
  17. 蛋白质湿转移印迹仪(Bio-Rad Laboratories,型号:Mini Trans-Blot®)
  18. 冰箱冷冻箱(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:10ECEETSA)
  19. SDS-PAGE电泳装置(Bio-Rad Laboratories,型号:Mini-Protean Tetra Cell)
  20. 标准轨道摇床(VWR,型号:1000型)
  21. 热循环仪(Thermo Fisher Scientific,Applied Biosystems TM,型号:Veriti TM 96孔)

软件

  1. Google Chrome(Google)或其他网络浏览软件
  2. Snapgene(Snapgene)或其他质粒浏览软件
  3. Image Studio Lite(LI-COR)或其他凝胶分析软件
  4. Axiovision(卡尔蔡司显微镜)或其他细胞成像软件
  5. ImageJ(由Wayne Rasband开发)或其他图像量化软件
  6. GraphPad Prism(GraphPad Software Inc.)或其他图形和统计分析软件

程序

  1. 生成用于C端标记的基因特异性Cas9 / sgRNA质粒
    1. 基因组序列检索
      1. www.toxodb.org ,导航到感兴趣的基因(GOI)的基因页面(图2A) 。通常在RH TIR1-3FLAG生产线上使用GT1序列。点击基因ID上方的“下载基因”链接(图2A)。
      2. 从ToxoDB.org上的“下载基因”页面,通过选择以下选项下载3'标记区域:选择一个报告:FASTA;序列类型:基因组;地区:翻译站点-80,翻译站点+200;下载类型:在浏览器中显示。选择“获取序列”链接(图2B)。
      3. 从序列检索页面,选择没有FASTA标识符的基因组序列并复制到剪贴板(图2C)。
    2. Protospacer选择
      1. 从真核病原体CRISPR指南RNA / DNA设计工具网页(EuPaGDT, http://grna.ctegd.uga.edu / ),首先输入“工作名称”。使用SpCas9建议的默认设置:'20 nt gRNA NGG PAM'。在“基因组选择”选项卡下,选择 T。 gondii血统和数据库(例如, T。gondii GT1 ToxoDB-32)。粘贴从ToxoDB.org复制的基因组序列,并选择“获取指导RNA”(图2D)。
      2. http://grna.ctegd.uga.edu/ 上的gRNA输出页面,选择< ( eg ,STOP-N ... N)下游的NGG PAM 的20nt gRNA原型间隔区序列<100> GATCGACTTAGTAACGCATC
    3. pSAG1 :: Cas9-U6 :: sgUPRT的突变( Addgene#54467 )(Shen等人,2014)
      1. 质粒pSAG1 :: Cas9-U6 :: sgUPRT瞬时表达SpCas9-HA-GFP核酸酶和单一指导RNA(sgRNA),其结合Cas9并指导其在TgUPRT基因中进行双链断裂。 Cas9 / sgRNA核酶的基因靶标特异性基于sgRNA的5'端的20nt原型空间序列。要将Cas9目标从 TgUPRT 切换到另一个基因,需要将20 nt原空间序列更改为步骤A2“原型空间选择”中设计的序列。使用Q5定点诱变(新英格兰生物实验室)突变pSAG1 :: Cas9-U6 :: sgUPRT,将20nt TgUPRT原型间隔区改变为针对感兴趣的基因选择的20nt原型间隔区(图2E )。标准的25μl反应包括:
        1μl(1ng)pSAG1 :: Cas9-U6 :: sgUPRT
        1.25μl(10μM)反向引物:(5'-AACTTGACATCCCCATTTAC)
        1.25μl(10μM)正向引物:
        (5' - (G)NNNNNNNNNNNNNNNNNNNN GTTTTAGAGCTAGAAATAGC) 9μl去离子H 2 O
        12.5μl2x Q5热启动PCR混合物
      2. Q5诱变热循环
        步骤1.在98℃变性30秒
        步骤2. 98℃10秒变性
        步骤3.在55℃30秒退火
        步骤4.在72℃延伸5分钟
        第5步。重复步骤2-4 24次
        步骤6.在72℃延伸2分钟
        第7步。保持在4°C直到进一步使用。
      3. 在含有1kb DNA梯的0.8%琼脂糖凝胶(TAE或TBE)上分解1μlPCR反应以检测扩增。预期的扩增子大小是9.68 kb。
      4. 激酶,连接酶,消化(KLD)反应(包括在Q5定点突变试剂盒中)
        1μlPCR产物
        3μl去离子H 2 O
        5微升2x KLD缓冲液
        1μl10x KLD酶混合物

        在室温下混合并孵育5分钟
      5. 转化NEB5α化学胜任的E。用1μl的KLD反应培养大肠杆菌。在冰上孵育30分钟。在42℃加热化学转化的细胞30秒。添加950μLSOC培养基(新英格兰生物实验室)到转化细胞和孵化在37°C混合1小时。传播100微升转化的E。在含有100μg/ ml氨苄青霉素的LB琼脂平板上培养大肠杆菌。 37°C孵育板过夜。第二天,从每个LB琼脂平板上挑取2-3个菌落,在5ml LB培养液+100μg/ ml氨苄青霉素中过夜培养,用于质粒提取。

      6. 使用台式离心机在3,000g x g下过夜培养细菌培养物10分钟
      7. 用质粒提取试剂盒(Macherey-Nagel)从细菌沉淀中提取质粒DNA。预热灭菌过滤缓冲液EB至70°C。
        用50μl预热的无菌过滤缓冲液EB将DNA洗脱到无菌的1.7ml微量离心管中
      8. 通过分光光度法(260nm / 280nm处的吸光度,NanoDrop(Thermo Fisher))测量DNA浓度和纯度。预期产量约为500-1000 ng /μl。
      9. 通过使用M13反向通用引物(5'-ACAGGAAACAGCTATGAC)(Genewiz)的Sanger测序确认pSAG1 :: Cas9-U6 :: sg“GOI”的突变的原型空间序列。


        图2. C末端mAID标签构建体的产生。A-E。生成基因特异性Cas9 / sgRNA质粒。来自ToxoDB.org的TgPKG(TGGT1_311360; C端标记的示例基因)的基因页面。红色箭头指向下载基因链接以获取所需的TgPKG基因组序列。 B.从ToxoDB.org上下载 TgPKG 的“下载基因”页面。红色星号突出显示需要输入的字段,如图所示。红色箭头指向获取序列链接。 C.ToxoDB.org的FASTA基因组序列(来自翻译终止密码子的-80至+200)(其为来自ToxoDB.org的TgPKG(-80至+200)),其为C端标签所需的gRNA和供体同源性设计所需。 D.从EuPaGDT( http://grna.ctegd.uga.edu/)指导RNA设计工具(裁剪以适合) )。红色的星号表示需要完成的基本领域。红色箭头指向“获取指南RNA”链接。 E.来自EuPaGDT的每条链上的TgPKG基因组序列(来自翻译终止密码子的-80至+200)的gRNA原始空间的输出(上方示意图)。选择具有来自EuPaGDT的质量评分的gRNA protospacer和protospacer毗邻基序(PAM)序列(表格)。通过定点诱变(下示意图)将pSAG1 :: Cas9-U6 :: sgUPRT中的TgUPRT原始间隔子与设计的TgPKG原始间隔子交换。 F.产生TgPKG特异性C-末端mAID-3HA,HXGPRT标签盒。来自ToxoDB.org的FASTA基因组序列(TgPKG)(来自翻译终止密码子的-80至+200),具有所示的突出特征。 mAID-3HA的PCR扩增,来自pYFP-mAID-3HA的HXGPRT标签盒,具有供体同源性(40bp)的5'和3'区域的Floxed HXGPRT到TgPKG(侧接Cas9断裂位点)
        分别添加到正向和反向引物(星号)
  2. 生成基因特异性AID标签盒
    1. Microhomology侧面选择和引物设计
      1. 5'微同源性侧翼是紧接在翻译终止密码子之前的感兴趣基因的最后40个核苷酸。 3'microhomology侧翼将是紧接着选定protospacer / PAM网站的第一个40 nt。通过使用约60个核苷酸的DNA寡聚引物进行PCR,这些侧翼将被附加到(m)AID标签盒(图2F)。
      2. 订购标签引物。标记正向引物将是40nt 5'同源侧翼+ GCTAGCAAGGGCTCGGGC。标签反向引物将是40nt 3'同源性侧翼(反向互补)+ ATAGGGCGAATTGGAGCTCC。我们从Integrated DNA Technologies( www.idtdna.com )购买25 nmole DNA寡核苷酸引物,进行“标准脱盐” 。
    2. PCR扩增和清理AID标签扩增子
      1. Q5 PCR设置。使用pYFP-mAID-3HA,Floxed HXGPRT( Addgene#87259 )(Brown ,2017)或pYFP-AID-3HA,Floxed HXGPRT( Addgene#87260 (Long等人,2017)质粒作为模板(图2F)。标准的500μl反应包括:
        100μl5x Q5缓冲液
        10μldNTPs
        5μL(50μM)标签正向引物(图2F)
        5μL(50μM)标签反向引物(图2F)
        5微升(5毫微克)质粒模板
        5μlQ5 DNA聚合酶
        370μl去离子H 2 O
        将PCR混合物分成5个PCR管,每个100μl
      2. Q5 PCR热循环。
        步骤1.在98℃变性30秒
        步骤2. 98℃变性10秒
        步骤3.在60℃退火30秒
        步骤4.在72℃延伸2.5分钟
        第5步。重复步骤2-4 29次
        步骤6.在72℃延伸2.5分钟
        第7步。保持在4°C,直到进一步使用
      3. 检查PCR质量。在含有1kb DNA梯的0.8%琼脂糖凝胶(TAE或TBE)上解决1μlPCR反应以检查扩增。 (m)AID-3HA,Floxed HXGPRT-3'同源性侧翼(预期的扩增子大小:mAID为2.9kb,AID为3.4kb)。
        标签扩增子由5'同源性侧翼接头 -
      4. 用PCR Cleanup Kit(Macherey-Nagel)进行PCR清除。合并5个PCR反应物并添加1,000μl缓冲液NTI。加750μl混合物到DNA柱中。用微量离心机以11000×g离心1分钟。放弃流量。将剩余的750μl混合物装入柱中。在11,000×g下离心1分钟。放弃流量。用700μl缓冲液A3清洗柱子。在11,000×g下离心1分钟。放弃流量。重复洗涤步骤并丢弃流过。再以11,000×g克离心另外3分钟。预热灭菌过滤缓冲液EB至70°C。
        用30μl预热的无菌过滤缓冲液EB将DNA洗脱到无菌的1.7ml微量离心管中
      5. 通过分光光度法(260nm / 280nm处的吸光度,NanoDrop(Thermo Fisher))测量DNA浓度和纯度。旨在为寄生虫转染至少有10微米的标签盒。重复PCR和清理步骤,如果需要更多的DNA。

  3. 细胞培养
    1. 人包皮成纤维细胞(HFF)培养
      1. 起动器文化。在35ml D10培养基(见配方)中,在37℃5%CO 2培养箱中的无菌T-175培养瓶中培养起始HFF细胞。
      2. HFF通道。一旦融合,取出D10培养基并用35ml无菌1x PBS洗涤并丢弃洗涤液。加入5毫升胰蛋白酶,替换盖帽,孵育细胞在37°C 1-5分钟,直到细胞相互分离(如使用10倍放大倍率检测)。牢牢地轻敲瓶子的侧面几次,使塑料脱离细胞。添加175毫升新鲜的D10培养基胰蛋白酶细胞和混合产生1:5细胞悬液。通过35毫升到新的T - 175。通过5毫升进入T-25s,使HFF饲养瓶支持弓形虫培养。通过96孔板每孔200μl和24孔板每孔1ml用于转染后的寄生虫克隆。
    2. 寄生虫文化
      1. 寄生虫信息。 T。弓形虫RH TIR1-3FLAG(基因型:RHΔ hxgprt Δku80 ; TUB1 :TIR1-3FLAG,SAG1 <因为它为AID系统提供了三个关键要素:△KU80 限制非同源结束连接,减少了AID标记的随机集成卡带; Δhxgprt基于HXGPRT介导的霉酚酸抗性提供了正确选择AID标签盒的正确遗传背景; TIR1-3FLAG是植物生长素受体在生长素诱导的SCF TIR1-靶向蛋白酶体降解标记蛋白质中所需的植物生长素受体。
      2. 寄生虫通道。用RH TIR1-3FLAG寄生虫接种融合的HFF T-25饲养瓶,每两天使用350μl新鲜寄生的寄生虫,或根据需要在自然出口后立即接种。在D10培养基中培养寄生虫在37℃5%CO 2中。

  4. (m)使用CRISPR基因组编辑(例如,TgPKG-mAID-3HA;图3)的AID标记;
    1. 寄生虫转染
      1. 培养寄生虫用于转染。在足够RH TIR1-3FLAG寄生虫(95μl新鲜从T-25寄生的寄生虫)中感染在T-25烧瓶中生长的融合宿主细胞以在感染后2天实现约75%宿主细胞裂解。刮单层和通过细胞悬液通过一个22 G钝头针3-5次破坏宿主细胞和释放所有寄生虫。避免使用针棒,并将使用过的针头小心处理到生物危害锐利容器中。预先湿润的3.0μm聚碳酸酯膜通过用10ml注射器将5ml HHE通过过滤器进入废物容器。将注射器溶解的寄生虫通过具有10ml注射器的过滤器放入50ml聚苯乙烯锥形小瓶中。通过过滤器再加入5毫升HHE到50毫升圆锥形中,以从过滤器收集额外的寄生虫。用台式离心机在18℃下离心过滤的培养物10分钟,在10ml HHE中重悬沉淀,用血细胞计数器计数细胞密度,并在相同条件下再次离心。随后,在Cytomix缓冲液中重新悬浮寄生虫细胞沉淀物(参见食谱)以达到4×10 7 ml -1的寄生物密度。
      2. 准备在电穿孔比色皿
        转染混合物 Cytomix缓冲液中有250μl寄生虫。
        10微克GOI特异性CRISPR / Cas9质粒(图2A-2E)
        10μgGOI特异性AID标签PCR扩增子(图2F)
        3μlATP(0.2M,100x)
        3μl谷胱甘肽(0.5M,100x)
        用Cytomix缓冲液调整至300μl。
      3. 电穿孔。 Electroporate寄生虫根据electroporator制造商的指示。我们使用带有4 mm间隙比色杯的BTX ECM-830电穿孔仪,具有以下参数:
        1,700 V
        176微秒的脉冲长度
        2个脉冲
        脉冲间隔100毫秒
        在电穿孔后立即将寄生虫转移到具有融合宿主细胞的新鲜T-25烧瓶中,并在37℃,5%CO 2下生长。
    2. 药物选择和寄生虫克隆
      1. 药物选择。在电穿孔和恢复后24小时,使用补充有黄嘌呤(50μg/ ml)的霉酚酸(25μg/ ml)开始用于(m)AID-3HA,Floxed HXGPRT构建体的药物选择。重复传代,直到耐药池变得稳定(通常2-3次直到培养稳定(即每隔两天用350μl接种新鲜出壳的寄生虫溶解单层) >
      2. 通过有限稀释克隆寄生虫。如上所述,在耐药群体的自然出口之后,净化并计数寄生虫。用D10培养基(无药物)稀释纯化的寄生虫以达到3个寄生虫/150μl。随后,将150μl/孔的这种稀释的培养物加入96孔板中,使融合的宿主细胞在37℃,5%CO 2下生长7天,而不移动以允许斑块通过溶解寄生虫复制来宏观地破坏宿主细胞单层)来发展。
    3. 筛选克隆用于基因重组
      1. 识别克隆。在斑块发育后,在倒置相差显微镜(例如Nikon TS100)下用肉眼检查96孔板的每个孔并寻找只含有一个斑块的孔(参见Ufermann等人 >,2017年为高分辨率斑块形态的例子)。将阳性寄生虫的寄生虫转移到含有融合的宿主细胞单层的24孔板中,并培养它们直到它们自然流出。
      2. 使裂解物用于诊断性PCR。从每孔接种一小份(例如,10μl)到含有汇合单层宿主细胞的新的24孔板中以继续培养寄生虫。其余的裂解培养物将用于诊断性PCR。添加1毫升寄生虫克隆到1.7毫升microfuge管。对于阴性对照,加入1毫升外出的野生型寄生虫(亲本系)到一个单独的1.7毫升微量离心管中。在18℃下用微量离心机将所述寄生物悬浮液沉淀在400μg×10分钟并除去寄生虫沉淀物的上清液。向每个沉淀中加入100μl在1x PBS中的蛋白酶K(PK)(0.2mg / ml)并混合。在37℃加热10分钟,50℃10分钟,98℃加热10分钟。
        冻结样品或在冰上放寒,然后进行诊断性PCR。
      3. 诊断PCR设置。应该执行两个简单的PCR来确定标记成功。 PCR1应专门扩增未修饰的基因座,而PCR2应专门扩增标记的基因座(图3A)。每25μl诊断PCR反应包括:
        2.5μl10x Taq缓冲液
        0.5μldNTPs

        0.25μl(50μM)正向引物(与PCR1和PCR2相同) 0.25μl(50μM)反向引物(PCR1和PCR2专用)
        1μlPK处理的寄生虫裂解物
        0.25μlTaq DNA聚合酶(New England Biolabs)
        20.25μl去离子H 2 O
        PCR1和PCR2的正向引物应该在翻译终止密码子的上游(5'侧)〜350bp处引发。 PCR1反向引物应在翻译终止密码子下游的3'UTR,〜300bp处引发。
        PCR2反向引物应该从mAID或AID序列开始〜+ 50
      4. 诊断PCR热循环
        步骤1.在98℃变性30秒
        步骤2. 98℃10秒变性
        步骤3.在60℃30秒退火
        步骤4.在68℃延伸1分钟
        第5步。重复步骤2-4 29次
        步骤6.在68℃延伸1分钟
        第7步。保持在4°C,直到进一步使用
      5. 解决诊断PCR扩增。在含有1kb DNA梯的1%琼脂糖凝胶(TAE或TBE)上分解10μlPCR反应以检查阳性扩增(图3C)。对于野生型未修饰的基因座,PCR1应产生约650bp的扩增子。对于成功标记的克隆,PCR2应产生约400bp的扩增子。使用抗HA抗体,可以通过其他方式进一步检测PCR阳性克隆,如免疫荧光显微镜(图3D)或Western印迹(图3E)(m)AID-3HA表位标签。
      6. 亚克隆标记的克隆。一旦鉴定出阳性克隆并确认,应将其从24孔板转移到T-25瓶中并冷冻保存以供将来使用。


        图3.(m)AID蛋白融合的产生和调节。 A.在RH TIR1-3FLAG系中CRISPR介导的C-末端mAID标记。此处显示的实例是编码蛋白激酶G(TgPKG)的基因,其经许可从[Brown等人,2017年]中转载)。注意诊断性PCR 1和2的位置。B. TgPKG-mAID-3HA表达的示意图,显示了从转录物上的备选翻译起始位点产生的两种PKG-mAID同种型。 IAA的加入促进了两种PKG-mAID同种型的同时降解。光盘。数据根据(Brown等人,2017年)修改的空间限制进行复制。 C.来自基因组DNA样品的诊断性PCR 1和2,显示mAID标签构建体成功整合入TgPKG(参见A的PCR位置)。 WT(野生型),TIR1-3FLAG亲本;标签,PKG I,II -mAID-3HA寄生虫。 D.PKG-mAID-3HA同种型I和II的免疫荧光用抗-HA mAb染色并用Alexa Fluor 488-缀合的二抗检测。比例尺= 5微米。 E.用识别HA(绿色)和醛缩酶(红色)的抗体探测裂解的PKG1,II-mAID-3HA寄生虫的蛋白质印迹分析。在裂解之前,用500μMIAA或载体(EtOH)处理寄生虫4小时。

  5. AID标签蛋白的消耗
    1. 寄生虫文化。用RH TIR1-3FLAG和RH POI-(m)AID-3HA寄生虫感染HFF培养物。用于感染的HFF单层的类型(例如,盘,孔板,烧瓶)和数量将取决于实验,但是对于生长素和媒介物处理,每寄生生产线至少需要两种培养物。 >
    2. 生长素治疗。使用1:1,000的生长素消耗终浓度为500μM的(m)AID标签蛋白。模拟处理由等体积的100%EtOH组成,终浓度为0.0789%(w / v)。在37°C孵育。实现完全蛋白质敲除所需的孵育时间将根据蛋白质定位,稳定性和丰度而变化。仅使用15分钟就可以完全敲除可溶性胞质(m)AID融合物,如YFP(Brown等人,2017; Long等人, ,2017)。在敲除后,寄生虫可直接检查表型,收获后续检测,或溶解(m)AID标记的蛋白水平的分析。
    3. 检测AID标签蛋白水平。 (m)AID构建体的3HA部分可以用于通过免疫荧光显微术(图3D)或Western印迹(图3E)使用抗HA抗体(图2)进行蛋白质检测。

数据分析

可以使用ImageJ密度测定软件进行(m)AID融合敲减的Western印迹数据的定量三次重复。我们建议您查看由圣何塞州立大学Dr. Luke Miller创建的在线资源( http://lukemiller.org/index.php/2010/11/analyzing-gels-and-western-blots-with-image-j/ )for ImageJ密度测量的详细指南。

  1. 在ImageJ中,打开(m)AID-3HA抗HA蛋白质印迹的灰度TIF图像。文件 - &gt;打开 - &gt; .tif
  2. 使用矩形工具,在第一条泳道的蛋白质条带周围绘制一个垂直矩形。
    这个矩形必须比它宽。
  3. 按Ctrl + 1选择第一个车道。一个'1'将出现在矩形。
  4. 左键单击矩形并将其拖到下一个通道。
  5. 按下Ctrl + 2选择第二个车道。 “2”将出现在第二个矩形。
  6. 左键单击最新的矩形并移至第三个通道。重复步骤5和6,直到每个车道都有一个矩形。
  7. 按下Ctrl + 3将数据绘制在直方图中。一个新的窗口会弹出。
  8. 使用线条绘制工具,在连接左侧背景线和右侧背景线的每个峰下绘制一条线。
  9. 在所有的背景线绘制完毕后,使用“魔棒”工具点击每个顶点的内部,从顶部(第1道)开始,向下工作。
  10. 每个峰(带)的密度值将在新的弹出表中找到。
  11. 重复步骤1-11加载控制污点。
  12. 将每个样品的总蛋白(AID-3HA密度/加样控制密度)归一化,然后将该比例转换为未处理对照的百分比(归一化样品值/归一化对照比率×100)。
  13. 根据制造商的说明,使用GraphPad Prism软件可以对来自一式三份实验的数据取平均值和分析统计学显着性。

笔记

T.弓形虫RH TIR1-3FLAG(基因型:RHΔ hxgprt Δku80 ; TUB1 : TIR1-3FLAG,SAG1 : CAT )(Brown et al。,2017; Long et al。,2017)提交给ATCC进行分发。将生长素degron系统应用于其他的 T。 gondii株,质粒pTUB1:OsTIR1-3FLAG,SAG1:CAT( Addgene#87258 )可用于转染。请访问 http://www.addgene.org/David_Sibley 获取其他与AID相关的质粒。

食谱

  1. 10倍Tris-乙酸乙二醇酯(TAE)缓冲液
    48.4克Tris碱
    11.42毫升冰醋酸
    20毫升0.5 M EDTA pH 8.0
    适量用去离子H 2 O至1L 在室温下储存
  2. 10倍Tris-Borate-EDTA(TBE)缓冲液
    108克Tris基地
    55克硼酸
    40毫升0.5 M EDTA pH 8.0
    适量用去离子H 2 O至1L 在室温下储存
  3. 10倍SDS-PAGE运行缓冲区
    30克Tris基地
    144克甘氨酸
    10克SDS
    适量用去离子H 2 O至1L 在室温下储存
  4. 10倍蛋白转移缓冲液
    30.3克Tris碱
    144克甘氨酸
    适量用去离子H 2 O至1L
  5. 1x蛋白转移缓冲液
    700毫升去离子H 2 O
    200毫升甲醇
    100毫升10X蛋白转移缓冲液
  6. 10倍磷酸盐缓冲盐水(PBS)
    2克KCl
    80克NaCl
    14.4克Na 2 HPO 4 4 2克KH 2 PO 4 4克/克 适量用去离子H 2 O至1L 在室温下储存
  7. 磷酸盐缓冲盐水+ Tween-20(PBST)
    899毫升去离子H 2 O
    100毫升10倍PBS
    1毫升吐温-20
    在室温下储存
  8. PCR裂解缓冲液
    蛋白酶K在PBS中0.2mg / ml
  9. D10中等
    1包DMEM粉剂
    3.7克碳酸氢钠
    2.38克HEPES
    10毫升的200毫米L-谷氨酰胺
    1毫升10毫克/毫升庆大霉素
    100毫升特征胎牛血清
    适量用去离子H 2 O至1L 用1升Stericup过滤器过滤灭菌,并在4°C储存
  10. Hank平衡盐溶液与HEPES和EGTA(HHE)
    含有10mM HEPES和1mM EGTA的Hanks平衡盐溶液
  11. 0.1M KPO 4缓冲液pH 7.6(用于Cytomix缓冲液)
    17.4克K 2 HPO 4 4 13.6克KH 2 PO 4 4 适量用去离子H 2 O至1L
  12. Cytomix电穿孔缓冲液pH值7.6(Soldati和Boothroyd,1993)
    100ml 0.1M KPO 4 pH 7.6 8.95克KCl
    22.1毫克氯化钙。
    1.02克MgCl 2 2/2 5.96克HEPES
    744毫克EDTA
    适量用去离子H 2 O至1L 无菌过滤器和存储在4°C
  13. 500毫摩尔3-吲哚乙酸(IAA /生长素)(1,000x库存)
    87.59mg 3-吲哚乙酸加入到1ml 100%EtOH中 在-20°C储存,避光

致谢

我们要感谢Nisha Philip博士和Andy Waters博士在“弓形虫”中为AID协议的开发提供有益的讨论和共享质粒。我们没有利益冲突要申报。

参考

  1. Brown,K. M.,Long,S.和Sibley,L. D.(2017)。 N-酰化的质膜结合决定了弓形虫的PKG功能 。 MBio 8(3)。
  2. Doench,J.G.,Hartenian,E.,Graham,D.B.,Tothova,Z.,Hegde,M.,Smith,I.,Sullender,M.,Ebert,B.L.,Xavier,R.J。和Root D.E。(2014)。 合理设计用于CRISPR-Cas9介导的基因失活的高活性sgRNA。 > Nat Biotechnol 32(12):1263-7。
  3. Kreidenweiss,A.,Hopkins,A.V.和Mordmuller,B。(2013)。 2A和基于生长素的degron系统促进恶性疟原虫(Plasmodium falciparum)中的蛋白质水平的控制( PLoS One 8(11):e78661。
  4. Long,S.,Anthony,B.,Drewry,L.和Sibley,L. D.(2017a)。 含有保守锚蛋白重复序列的蛋白调控弓形体稳定性,运动性和细胞入侵弓形虫。 Nat Commun 8(1):2236.
  5. Long,S.,Brown,K.M.,Drewry,L.L.,Anthony,B.,Phan,I.Q.H。和Sibley,L.D。(2017b)。 钙调蛋白样蛋白定位于圆锥体调节动力和细胞侵袭 弓形虫
  6. Nishimura,K.,Fukagawa,T.,Takisawa,H.,Kakimoto,T。和Kanemaki,M。(2009)。 一种用于非植物细胞中蛋白质快速消耗的基于生长素的degron系统 Nat Methods 6(12):917-922。
  7. Philip,N.和Waters,A.P。(2015)。 疟原虫的条件降解钙调磷酸酶揭示寄生虫定殖的主机和载体。细胞宿主微生物 18(1):122-31。
  8. Shen,B.,Brown,K.,Long,S.和Sibley,L. D.(2017)。 开发CRISPR / Cas9用于弓形虫高效基因组编辑 。方法Mol Biol 1498:79-103。
  9. Shen,B.,Brown,K.M.,Lee,T.D。和Sibley,L.D。(2014)。 使用CRISPR / Cas9对不同弓形虫株进行高效基因破坏。 MBio 5(3):e01114-01114。
  10. Sidik,S.M.,Hackett,C.G.,Tran,F.,Westwood,N.J。和Lourido,S。(2014)。 使用CRISPR / Cas9的弓形虫抗体的有效基因组工程 PLoS One 9(6):e100450。
  11. Soldati,D.和Boothroyd,J.C。(1993)。 专性细胞内寄生虫瞬时转染和表达弓形虫 科学 260(5106):349-352。
  12. Starnes,G.L.,Coincon,M.,Sygusch,J。和Sibley,L.D。(2009)。 醛缩酶对寄主入侵宿主期间的能量产生和桥接粘附素 - 肌动蛋白细胞骨架相互作用是必不可少的细胞宿主微生物 5:353-364。
  13. Teale,W. D.,Paponov,I. A.和Palme,K.(2006)。 生长素的行动:发信号,运输和控制植物生长发育。
  14. Ufermann,C-M。,Muller,F。,Frohnecke,N.,Laue,M。和Seeber,F。(2017)。 弓形虫噬菌斑试验:改善超微结构和定量评估溶解性寄生虫的增长。 Parasitol 180:19-26。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Brown, K. M., Long, S. and Sibley, L. D. (2018). Conditional Knockdown of Proteins Using Auxin-inducible Degron (AID) Fusions in Toxoplasma gondii. Bio-protocol 8(4): e2728. DOI: 10.21769/BioProtoc.2728.
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